Multiple sclerosis (MS) is a chronic, inflammatory neurodegenerative disease that affects the central nervous system and may lead to severe disability within 10-15 years. MS is diagnosed by clinical symptoms and presence of inflammation detected by magnetic resonance imaging. Other diseases present with similar symptoms, so diagnosis of MS is challenging. The lack of clear and objective biomarkers for diagnosis, monitoring, or prediction of outcomes complicates MS detection and treatment of MS patients. We propose to use a novel assay developed in our laboratory for analysis of DNA methylation to identify MS-specific changes of DNA methylation in multiple genes within each specimen, and to create a composite biomarker combining genes with the most diverse patterns of methylation. The blood-brain barrier is disrupted in MS patients, genomic DNA from dead cells is released into the bloodstream, so the assay can be based on cell-free circulating DNA. Specific Aims of the project are: (1) to establish a composite biomarker for detection of patients with relapsing-remitting MS in remission based on DNA methylation profile of cell-free circulating plasma DNA;(2) to establish a composite biomarker for detection of patients with relapsing-remitting MS during exacerbations based on DNA methylation profile of cell-free circulating plasma DNA, and to compare it with the biomarker established in Aim 1;(3) to compare DNA methylation profiles of patients with relapsing- remitting MS and secondary-progressive MS and to determine biomarkers specific for progression. The assay allows simultaneous detection of methylation in 56 gene promoters using DNA from 0.15 ml of plasma. The assay successfully detected MS-specific changes in DNA from plasma of MS patients. Completion of this project will produce a novel set of objective observer-independent biomarkers based on a fast, simple, and inexpensive procedure, and utilizing a simple, safe, and minimally invasive specimen collection. There are no reports on MS-specific DNA methylation, so this project will be the first to address this deficiency. The utility of cell-free circulating DNA for testing in MS has never been reported, so this project will be the first to use this opportunity. Consequently, other components of this project are without precedent: a biomarker for different forms of RR-MS, a biomarker for prediction of an attack, and a biomarker for disease progression to SP-MS will be developed for the first time. Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system, which can lead to severe disability and death. Diagnosis of MS is difficult because initial stages of the disease do not have obvious symptoms or even any symptoms at all, and there are no objective markers specific for MS. In this project we will use a new technique developed in our laboratory to evaluate DNA modifications (methylation) in blood of MS patients with active MS (attack or exacerbation) and stable MS (remission). Completed project will provide the first objective biomarker based on MS-specific DNA methylation, which can be used for diagnosis of MS, for evaluation of treatments success, and for prediction of outcomes.